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Large scale purification of recombinant bovine deoxyhypusine hydroxylase
Author(s) -
Smith Joshua L,
Wang Yinglu,
Huang JenqKuen,
Wen Lisa
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.625.5
Subject(s) - affinity chromatography , biochemistry , biology , agarose , enzyme , fusion protein , protein purification , recombinant dna , microbiology and biotechnology , gene
The mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine. Hypusine synthesis is a posttranslational modification catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) which is essential for the function of eIF5A in cell proliferation and survival. It has been suggested that posttranslational modifications of eIF5A could be a suitable target for anticancer therapy. A detailed knowledge of structure‐function relationship of either enzyme will help in designing better inhibitors thus better control of proliferative diseases. Recently, we have cloned and expressed functional bovine DOHH (bDOHH) (Huang et al., Protein Expr. Purif. 54, 126–133, 2007). Here we report large scale purification of bDOHH. The GST‐tagged DOHH fusion protein was overexpressed by IPTG induction and the fusion protein purified by affinity chromatography with a glutathione agarose column. The GST tag was removed by thrombin digestion. The bDOHH protein was then separated from the GST carrier by glutathione agarose. The monomic bDOHH protein was separated from aggregates by gel filtration on a Superdex‐200 column. The purified protein is being used for initial crystallization screening. Supported by grants from National Institutes of Health 1R15 GM60266‐01A1 and the University Research Council at WIU.